Tumor Necrosis Factor (TNF-α) and Lymphotoxin (TNF-β) (hereinafter, TNF refers to both TNF-α and TNF-β) are cytokines which have many effects on cells (Wallach, D. (1986) in: Interferon 7 (Ion Gresser, Ed.), pp. 90-124, Academic Press, London, and Beutler, B. and Cersmi, A. (1987) New England J. Med. 316:379-385). Both TNF-α and TNF-β initiate their effects by binding to specific cell surface receptors. Some of the effects are likely to be beneficial to the organism: they may destroy, for example, tumor cells or virus infected cells and augment antibacterial activities of granulocytes. In this way, TNF contributes to the defense of the organism against infectious agents and to recovery from injury. But, quite clearly, both TNF-α and TNF-β have also effects which can be extensively deleterious. There is evidence that over-production of TNF-α can play a major pathogenic role in several diseases. Thus, effects of TNF-α, primarily on the vasculature, are now known to be a major cause for symptoms of septic shock (Tracey, K. J. et al. (1986) Science 234:470-474). In some diseases, TNF may cause excessive loss of weight (cachexia) by suppressing activities of adipocytes and by causing anorexia and TNF-α was thus called cachectin. It was also described as a mediator of the damage to tissues in rheumatic diseases (Beutler, op. cit.) and as a major mediator of the damage observed in graft-versus-host reactions.
There is therefore a necessity in finding out ways to eliminate or antagonize endogenously formed or exogenously administered TNF. One attempt in this direction was the isolation from human urine of a first TNF Binding Protein called TBP-I and shown to be able to antagonize the effects of TNF. This antagonism was determined both by measuring reduction of the cytotoxic activity of TNF, as well as by measuring interference of TNF binding to its receptors.
The protein TBP-I was first described in our U.S. patent application Ser. No. 07/243,092 filed on Sep. 12, 1988, (now abandoned in favor of a continuation that issued as U.S. Pat. No. 5,595,953), in which was disclosed a process for its purification to homogeneity from human urine by chromatography on CM-SEPHAROSE followed by high performance liquid chromatography (HPLC) on MONO Q and MONO S columns and reversed-phase HPLC. The homogeneous TBP-I thus obtained had an apparent molecular weight of about 27,000 in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) under both reducing and non-reducing conditions. Homogeneity of the purified protein was confirmed by microsequence analysis which revealed a single N-terminal sequence: Asp-Ser-Val-Cys-Pro-(SEQ ID NO: 1).
TBP-I was shown to protect cells from TNF toxicity at concentrations of a few nanograms per ml and to interfere with the binding of both TNF-α and TNF-β to cells, when applied simultaneously with these cytokines. Further examination of the mechanism by which TBP-I functions revealed that TBP-I does not interact with the target cell, but rather blocks the function of TNF by binding TNF specifically, thus competing for TNF with the TNF receptor.
As a result of this finding, we attempted an alternative approach for the purification of TBP-I, whereby urinary proteins or fractions thereof were applied on a column of immobilized TNF and, after removal of unbound proteins, the proteins which bound to the column were eluted, in bioactive form, by a decrease of the pH. In SDS PAGE analysis, most of the protein in the eluate migrated as a single broad band with apparent molecular size of 30,000±2,000.
When applied to further fractionation by reversed-phase HPLC, the proteins eluting from the TNF col showed the presence of two active components: one, TBP-I, eluting as expected at 27% acetonitrile and, in addition, a second TNF-binding protein, eluting at a somewhat higher acetonitrile concentration (31%). This TNF-binding protein is new and is herein called TBP-II. Both proteins provide protection against the in vitro cytocidal effect of TNF and both bind TNF-β less effectively than TNF-α. Although in SDS PAGE analysis the two proteins, TBP-I and TBP-II, appeared to have a very similar molecular size, they could clearly be distinguished from each other by lack of immunological cross reactivity, differing N-terminal amino acid sequences and differing amino acid composition.